Leukocyte regulatory receptors (LRRs) effect a variety of immune functions, including self/nonself recognition and fine regulation of the response to infection. Membrane receptors belonging to several different LRR gene families are expressed by natural killer (NK) cells and detect a variety of ligands or absence thereof on the surfaces of different target cells. The full complement of receptors that mediate NK function is likely not understood. We have cloned the novel immune-type receptor (NITR) genes, which constitute a large multigene family encoding transmembrane immunoglobulin (Ig)-type molecules, from bony fish. NITRs exhibit structural characteristics of NK receptors and other LRRs, including activating/inhibitory-transmembrane signaling, and possess ectodomains of the Ig/T cell antigen receptor (TCR) variable (V) region type. NITR genes, which have been identified in several species of bony fish, including zebrafish (Danio rerio), do not undergo somatic reorganization and represent the only example of multigenic (cell surface) receptors outside of Ig and TCR genes in which highly diversified V regions likely are utilized as the core recognition elements of a receptor family. NITRs constitute a unique and potentially informative link between the innate and adaptive immune systems. Resolution of the complete zebrafish NITR gene cluster has identified multiple inhibitory forms and one activating form ofNITR. NITR V specificity for both endogenous and exogenous ligands will be examined using a number of different approaches based on: 1) soluble recombinant NITRs, 2) an NFAT-coupled GFP reporter assay for NITR ligand binding and 3) the expression of transgenic chimeric zebrafish/medaka (Oryzies latipes) Mhc I. A second model system, channel catfish (Ictalurus punctatus), in which NK-type cell lines that recognize allogenic targets have been derived, will be used for characterizing specific NITRtarget recognition. Information gained in the catfish system will be applied to identification of NITR-ligand interactions in zebrafish. The cell surface biochemistry of NITRs will be characterized and structural biology approaches will be used to compare NITR V regions to those of adaptive immune receptors. Knowledge of the full genomic structure of the zebrafish NITR cluster along with the relative ease of introducing transgenes and modulating expression levels of specific mRNAs in this species will be used to characterize both cell lineage- and developmental stage-specific expression of NITRs and to examine how NITR gene expression relates to innate immunity during early development. The unique advantages of zebrafish as a developmental model will permit us to examine vertebrate innate immunity prior to the initiation of adaptive immune function. Collectively, the multifaceted approach being utilized will define the essential structure/function relationships of a novel family of putative recognition molecules in a uniquely informative model system. Such information is essential to understanding the evolution and interrelatedness of NK and other LRR-mediated immune recognition processes, as well as the role of innate immunity during development. Understanding this relationship has broad health consequences in terms of mechanisms of resistance to infection and susceptibility to malignancy.